Clonal amplification by the polymerase chain reaction (PCR) is well-established. A sample is partitioned so that individual nucleic acid molecules within the sample are localized within many separate regions. This can be done in a variety of ways, including isolation in microwells, capillaries, chambers and emulsions. The partitioning of the sample allows one to control the number of different molecules by limiting dilution. For example, to ensure that the instance of two nucleic acid templates in a compartment is a low frequency event, the template is diluted so that, on average, there is less than one template molecule per compartment. As a result, each compartment will generally contain “0” or “1” molecules; after PCR, amplification is either negative or positive, respectively.
One approach to clonal amplification involves solid phase amplification at limiting template dilution in aqueous compartments (i.e. droplets) in an oil emulsion, wherein the compartments contain particles (e.g. beads), template, nucleotides and nucleic acid amplification enzyme. After PCR, some compartments contain clonally amplified beads, but only at 10-20% abundance (governed by a Poisson distribution). This presents an efficiency problem. While clonal amplification is achieved, there are simply too many negative reactions. If one wants to carry out a further reaction or analysis, one cannot efficiently perform this on all of the beads, since only 10-20% would have DNA present.
What is needed is a solution to this efficiency problem.